Project Summary ? Project 2 The programmatic goals of this PSOC application are to understand how the physical characteristics of tumor cells within their 3D microenvironment impact the metabolic changes that are required to maintain their malignant state. Project 2 focuses on a key outcome of these metabolic changes, specifically, the generation of microvesicles (MVs), a class of extracellular shed vesicles that are shed from the plasma membranes of cancer cells. MVs are receiving increasing attention because of their roles in various aspects of cancer progression, including the induction of changes within the tumor microenvironment, the stimulation of tumor angiogenesis, and contributions to the metastatic process. However, although we understand some of the biochemical signals that trigger MV production, we know little about the physical determinants that drive their formation along cancer cell surfaces, nor about how the physical microenvironment influences their biogenesis, and ultimately their functions. We intend to probe these important issues in Project 2, through three lines of experimental inquiry. 1) Determine the physical relationships governing MV biogenesis and size distribution. We will examine the physical changes in membrane shape and protein-protein interactions that result in membrane curvature and the maturation of MVs. In particular, we will take advantage of our recent discovery that glycans (e.g. Mucin 1), which are highly expressed in cancer cells, have a key role in MV biogenesis. 2) Determine the reciprocal relationship between the physical properties of the extracellular matrix and MV formation. While the vast majority of studies on cancer cell metabolism have been performed in 2D cell culture settings, we will explore how the physical features of the 3D microenvironment influence the metabolic cues that drive MV biogenesis. 3) Establish physical read-outs such as vesicle size as indicators of MV function in tumor-stroma and reciprocal stroma-tumor vesicle transfer. We will implement the necessary engineering platforms to isolate MVs from other classes of vesicles in order to eliminate the ambiguity that often exists regarding studies using heterogeneous extracellular vesicle preparations and thereby define more precisely the functions of MVs. We will determine the effects of MVs on surrounding non-cancerous cells (adipose stromal cells and endothelial cells), and examine whether this leads to the production of new MVs (e.g. from adipose stromal cells) that might feed-back regulate the metabolism and invasiveness of tumor cells. These studies will take advantage of the two PSOC Cores, Tissue Microfabrication and Biophysics and Metabolic Imaging, and will complement and benefit from Projects 1 and 3. Collectively, they will shed new light on cancer cell metabolism and how it gives rise to MV biogenesis, with the expectation being that ultimately novel therapeutic strategies will emerge from these efforts.